Identification, Cloning, Expression, and Characterization of the Gene for Plasmodium knowlesi Surface Protein Containing an Altered Thrombospondin Repeat Domain

Virginia Polytechnic Institute and State University, Блэксбург, Virginia, United States
Infection and Immunity (Impact Factor: 3.73). 10/2005; 73(9):5402-9. DOI: 10.1128/IAI.73.9.5402-5409.2005
Source: PubMed


Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion to host cells
are considered attractive vaccine targets. Aided by the availability of the partially completed genome sequence of the simian
malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA sequence that encodes a 262-amino-acid-long protein, containing an altered version of the
thrombospondin type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse functions,
such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a multistage antigen that is expressed
on the surface of P. knowlesi sporozoite and erythrocytic stage parasites. Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting that PkSPATR is a parasite ligand that could be involved in sporozoite
invasion of liver cells. Furthermore, recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that native PkSPATR is immunogenic during infection. Further efficacy evaluation studies
in the P. knowlesi-rhesus monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a vaccine
against human malarias.

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Available from: Sanjai Kumar
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    • "In fact, it was previously demonstrated that recombinant SPATR from Plasmodium falciparum and its orthologue in P. knowlesi were able to bind to HepG2 cells with high specificity, and antibodies generated against SPATR could inhibit P. falciparum sporozoite invasion into liver cells [6,7]. The P. falciparum SPATR protein was recognized by sera from naturally infected Africans but not control sera from two non-immune donors, indicating that this protein was recognized by the host immune system [6]. "
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    ABSTRACT: Background: Plasmodium knowlesi is the fifth species identified to cause malaria in humans and is often misdiagnosed as Plasmodium malariae due to morphological similarities. The development of an inexpensive, serological detection method utilizing antibodies specific to P. knowlesi would be a valuable tool for diagnosis. However, the identification of specific antigens for these parasites remains a major challenge for generating such assays. In this study, surface protein containing an altered thrombospondin repeat domain (SPATR) was selected as a potentially specific antigen from P. knowlesi. Its multistage expression by sporozoites, asexual erythrocytic forms and gametocytes, along with its possible role in liver cell invasion, suggests that SPATR could be used as a biomarker for diagnosis of P. knowlesi. Methods: The spatr gene from P. knowlesi was codon optimized and cloned (pkhspatr). Recombinant pkHSPATR protein was expressed, purified, and evaluated for its sensitivity and specificity in immunoblot and ELISA-based assays for detecting P. knowlesi infection. Results: The recombinant pkHSPATR protein allows sensitive detection of human P. knowlesi infection in serum samples by immunoblot and ELISA. Conclusions: With further research, recombinant pkHSPATR protein could be exploited as a marker for detection of P. knowlesi infection in humans. Therefore, this finding should contribute to the development of immunodiagnostic assays for the species-specific detection of malaria.
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